Had trouble with first post. Hope I didn't duplicate! LKM
----------------------------Original message----------------------------
Hi, Netters!
I hope someone can help me figure out where I'm going wrong. Here's the
problem: I am trying to propagate M13mp18T7 phage (carry the T7 RNA polymerase
gene) and I have not been able to get plaques!
My procedure (in brief) is to grow XL-1 blue bacteria in LB with tetracycline
at 37degreesC for 6-8 hours. The tet is to make sure all of the bacteria have
the F' episome. I have checked my incubator to make sure the temp is 37 and
it is. I innoculate top agar which has been equilibrated to 45C with 100ul
of phage (I use fresh dilutions in LB of my stock) and 100ul of bacterial
culture. Vortex and plate onto LB plates, incubate overnight at 37C. The
next day I have lovely lawns of bacteria, but nothing that resembles a plaque
in any of the plates. I even got a fresh phage stock from Invitrogen, but
it isn't working!!! What am I doing wrong? Perhaps I am getting plaques but
I don't recognize them? I have plated lambda and T4 before, and I know that
the M13 plaques should be turbid (not clear) but holding the plates up to
the light doesn't show me any slightly clearer spots. How big should they
be? Any help will be greatly appreciated. Please EMail me as well as post-
ing since our Net News connection is frequently wiped out before I get to
see my answers... Thanks. Laura
LKM100F at ODUVM.cc.odu.edu